Normal cells must coordinate the activities of a number of cellular processes and pathways. One of the most important mechanisms by which this is accomplished is transcriptional regulation. Microarray hybridization experiments have identified many transcripts that are dysregulated in response to a given challenge or disease (1, 2). The identity of these dysregulated transcripts often permits an insight into the molecular nature of the process in question. These transcripts may also serve as important diagnostic and/or prognostic markers.
Once specific disease markers have been identified, it is important to develop rapid and reliable tests for detecting them. The applications for such tests are wide ranging. They can be used in clinical laboratories, to guide treatment decisions, or they can be used remotely, for the rapid detection of biological weapon dissemination. Real-time PCR is a method ideally suited for this purpose because of its exquisite sensitivity and speed. However, while there are a number of methods for quantifying PCR in real-time, each suffers from limitations.
In the past, transcript quantification was accomplished with northern blots, which are time consuming and typically rely on radio-isotopic methods of detection. Northern blots provide information on the length of a mRNA as well as its abundance but are generally limited to the study of 1 or 2 different mRNAs. Dot blots allow slightly more mRNAs to be analyzed but do not provide any information about transcript length or integrity.
One problem associated with preparing probes for Northern, Southern and dot blot analysis is that the failure to remove unincorporated label can result in unacceptably high background signals. Labels can be removed via any one of a number of approaches but this is an extra step requiring fluid handling steps that are difficult to automate.
The more recently developed microarray technologies allow one to quantify thousands of different RNA species in parallel. One limitation of microarrays is that only 1 or 2 different samples may be analyzed per array. This limitation results from the number of compatible fluorescent labels that may be used in a single hybridization experiment and the hybridization characteristics of the features on the array.
The number of samples that can be processed for microarray analysis in a given amount of time is also limited. The sample must be denatured to allow primers to anneal and a reverse transcriptase must be added to synthesize cDNA. The sample must be reverse transcribed in the presence of a fluorescently-labeled deoxynucleobase triphosphate so that it can be visualized on the array. Prior to applying the cDNA to the array however, unincorporated fluorescent nucleotides must be removed from the sample. This process involves multiple fluid handling steps, is difficult to automate, and adds significantly to the cost of an experiment. These difficulties are not unique to microarray experiments as any hybridization based detection method requires the separation of unincorporated label prior to applying a hybridization probe. Failure to do so often results in unacceptable background signal.
Ideally, one would like to increase the power of transcript analysis by analyzing thousands of mRNAs in thousands of different samples. This can be accomplished by increasing the throughput of microarfay experiments or by extending real-time PCR techniques to permit the analysis of more transcripts in parallel. Real-time PCR is an attractive alternative to microarrays as it can be used to analyze a few genes in a large number of samples. However, quantifying PCR in real-time is challenging, expensive, and limited to a few genes at a time.
Currently, there are three major types of “probes” used for quantifying PCR in real-time. They include double stranded DNA (dsDNA) binding dyes, such as ethidium bromide (EtBr) (3); the Taqman™ probe (see U.S. Pat. No. 5,723,591); and molecular beacons™ such as described in U.S. Pat. No 6,037,130. While each have certain advantages, none are ideally suited for monitoring the progress of many nucleic acid amplification reactions in real-time. For example, dsDNA binding dyes enjoy a substantial increase in fluorescence upon binding to dsDNA and can be included in a PCR to monitor the appearance of dsDNA, presumably due to PCR amplification. Their popularity results from their ease of use in that no probe design or oligonucleotide synthesis is necessary. However, these dyes cannot distinguish amplicon dsDNA from template dsDNA. Although this is not a problem when minute amounts of template are used, it can become a problem when complex template preparations, such as cellular DNA and whole blood, are used or when considerable amounts of template DNA are required (e.g., allele specific PCR (3, 4)). If 500,000 cells served as the starting material (as might be required for the detection of rare polymorphisms) then there would be micrograms of DNA template in the PCR. dsDNA binding dyes will generate a strong signal in the presence of this amount of template DNA, irrespective of the progress of the PCR. Accordingly, they cannot be used to monitor the progress of the PCR reaction.
dsDNA binding dyes bind to DNA non-specifically; thus, no nucleotide specificity is obtained. One must perform a melting curve after the amplification reaction to verify that the amplicon has the expected melting temperature. In practice, the melting curve requirement adds time to the procedure and limits amplicon size to approximately 300 bp. Linearity of the fluorescence signal becomes compromised when amplicons greater than 300 bp are studied. Furthermore, temperatures that denature DNA are encountered during each cycle of the PCR. During the time that the DNA is denatured, no signal can be measured using dsDNA binding dyes. This restricts fluorescence measurements to a period of time when the temperature of the reaction mixture is less than the melting temperature of the amplicon of interest.
Moreover, since these dyes must be present at a significant concentration in the amplification reaction, they add to its cost. High concentrations of dye may also impair polymerase fidelity and processivity (5). Finally, because they are mutagenic, the cloned products of a PCR monitored with these dyes may not be faithful copies of the starting material.
The Taqman™ probe is a short oligonucleotide that contains a fluorophore and a quencher. The fluorophore emission is mitigated by the quencher via energy transfer. During PCR, the probe anneals to the amplicon and is hydrolyzed by the 5′ to 3′ exonuclease activity of the polymerase. An increase in fluorescence signal is inferred to be due to an increase in the amount of amplicon present.
Fluorescence resonance energy transfer (FRET) efficiency decreases with the inverse sixth power of the distance between the reporter and the quencher. In practice, this limits the distance between the reporter and the quencher to less than 70 Angstroms (Å). This is a serious drawback of the Taqman™ assay. On one hand one wishes to design a longer probe to enhance hybridization specificity. However, a long probe will not permit efficient quenching of the reporter molecule by the quencher. Derivatizing the oligonucleotides with minor groove binding antibiotics improves its hybridization properties without increasing probe lengths (6). However, such modifications are expensive and requires special synthetic techniques.
Additional constraints on Taqman™ probe design exist and are problematic. For example, the probes must contain a modified nucleotide at their 3′ terminus to prevent extension by the polymerase. Taqman™ probes also cannot have a guanosine residue at their 5′ terminus or the fluorophore will be excessively quenched even after the oligonucleotide has been hydrolyzed. Homopolymeric runs of greater than four guanosines in a row are also not allowed and no more than 2 of the final 5 bases may be guanosine.
Furthermore, to be compatible with Taqman™ probes, the DNA polymerase used in the amplification reaction must have a 5′ to 3′ exonuclease activity (7). Thermophilic DNA polymerases commonly used for PCR including Vent™, Pfu™, and Tfu™ lack this activity and are therefore incompatible with the use of Taqman™ probes. This is problematic because, unlike Taq, these polymerases have the desirable property of high fidelity amplification. Thus far, only Taq DNA polymerase has been used successfully with Taqman™ probes.
Finally, Taqman™ probes require thermal denaturation in order to detect an amplicon, and are not capable of monitoring isothermal amplification methods such as Rolling Circle Amplification (RCA), Nucleic Acid Sequence Based Amplification (NASBA) and 3SR (8–10). The products of the detection process, fluorescent nucleobase monophosphates, can diffuse from their site of synthesis and as such, the Taqman™ assay is unsuitable for in situ applications.
Regarding, molecular beacons™, such as those described in U.S. Pat. No. 6,037,130, like the Taqman™ probe, they also consists of an oligonucleotide derivatized with a fluorophore and a quencher. The molecular beacon™ probe has internal complementarity and forms a stem loop in the absence of a target sequence. Stem loop formation brings the fluorophore and quencher into proximity and reduces the fluorescence emission. In a PCR, the molecular beacon™ hybridizes to an amplicon and its fluorescence emission increases.
However, molecular beacons™ also have a number of problems associated with them, many of which are similar to those described above in connection with the Taqman™ probes. Like Taqman™ probes, the design of molecular beacon™ oligonucleotide probes remains challenging. They must have a region of internal complementarity that is stable enough to remain in a stem loop in the absence of an amplicon but not so stable that its hybridization to the amplicon is compromised. Efforts to increase the hybridization efficiency by increasing the concentration of molecular beacon™ probe will result in decreased amplification efficiency, since the DNA polymerase must displace hybridized beacons™ during the reaction which decreases the rate of polymerization (an excess of probe also increases background fluorescence). Only a few thermophilic DNA polymerases possess the strand displacement activity required to displace a hybridized molecular beacon™.
Furthermore, as the reaction temperature rises during the PCR, molecular beacon™ probes denature and the observed fluorescent signal increases independent of the progress of the amplification reaction. Like dsDNA binding dyes, this restricts fluorescence measurements to a period of time when the temperature of the reaction mixture is less than that of the molecularbeacon™ probe.
Finally, the hybridization of either a Taqman™ or molecular beacon™ oligonucleotide probe to the target amplicon is quite sensitive to the primary sequence of the amplicon. As a result, innocent polymorphisms near the region being interrogated by the probe can render the probe completely ineffective as a reporter of amplicon concentration. While dsDNA binding dyes do not suffer from this limitation, they are incompatible with the complex templates commonly encountered in clinical and research labs. The molecules of the present invention are the only type of real-time PCR probe that can overcome this problem.
Thus, there is a clear need for a reagent that not only addresses the above-noted problems but combines the sensitivity of oligonucleotide probes with the convenience of dsDNA binding dyes. This need will become increasingly apparent as more ambitious expression profiling projects are undertaken. For example, there is currently great interest in performing thousands of PCRs in parallel in small, isolated chambers. One might want to rapidly analyze transcript levels in a very small amount of a particular tissue source. This type of approach, in which several dozen genes are analyzed simultaneously, has shown great promise in the early detection of biological weapon exposure (11). While the instrumentation for performing thousands of PCRs in parallel exists, there are considerable problems associated with monitoring these reactions simultaneously. Also, baseline expression levels of the genes of interest can vary greatly, as can the window in which real-time quantification will be in the linear range.
A large scale, real-time PCR approach necessitates that each chamber receive an equal amount of template. This amount must be sufficiently large for rare transcripts to be represented. However, this poses a problem for the use of dsDNA binding dyes as probes of these reactions since they will non specifically bind to the template and generate a background signal. This problem is exaggerated when a rare transcript is analyzed because more template is required. Taqman™ probes and molecular beacons™ are also not acceptable for a large scale, real-time PCR based approach for a number of reasons, not the least of which is cost. Probe design for both is problematic and this experiment potentially requires thousands of quality controlled oligonucleotide probes. Even if probes could be successfully designed with 90% efficiency, there would still be hundreds of probes requiring re-design and re-synthesis.
With respect to biological weapon (BW) detection, it is essential that any detection method used be robust enough to detect those agents that have been designed precisely to make detection more difficult. For example, if a weapons producer knew the sequence of the Taqman™ or molecular beacon probe being used for detection, it would be trivial to modify the sequence of the BW agent such that it is no longer detectable. This leads to an ever escalating process of BW agent modification to avoid detection and new probe design and synthesis to detect the modified agent. The ultimate outcome of this process is that one must perform a test with hundreds and possibly thousands of probe combinations to be assured that a modified agent has not been used. This is especially problematic when purified template is in limiting supply, as it so often is.
Thus, while it is clear that various real-time approaches to expression profiling will be used in the future, it is equally clear that the existing methods of quantification are inadequate for the experiments currently under consideration. Each of the approaches to cDNA labeling and real-time PCR analysis described herein, while solving some of the problems associated with traditional methods, introduces several problems of its own. In general, most of these methods are expensive, require extensive sample preparation, and require extensive fluid handling steps to separate incorporated from unincorporated labels. Therefore, new methods of quantification that do not suffer from these limitations are needed.
In sum, a need currently exists for sensitive and inexpensive methods for labeling nucleic acids that 1) do not require the separation of unincorporated label prior to downstream applications, 2) can be used to monitor nucleic acid amplification in real-time and 3) can be used to detect the presence of a polymerase. The present invention provides a method and reagents for accomplishing these and other goals.